Systemic deregulation of autophagy upon loss of ALS- and FTD-linked C9orf72

Abstract

A genetic mutation in the C9orf72 gene causes the most common forms of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein, predicted to be a DENN-family protein, is reduced in ALS and FTD, but its functions remain poorly understood. Using a 3110043O21Rik/C9orf72 knockout mouse model, as well as cellular analysis, we have found that loss of C9orf72 causes alterations in the signaling states of central autophagy regulators. In particular, C9orf72 depletion leads to reduced activity of MTOR, a negative regulator of macroautophagy/autophagy, and concomitantly increased TFEB levels and nuclear translocation. Consistent with these alterations, cells exhibit enlarged lysosomal compartments and enhanced autophagic flux. Loss of the C9orf72 interaction partner SMCR8 results in similar phenotypes. Our findings suggest that C9orf72 functions as a potent negative regulator of autophagy, with a central role in coupling the cellular metabolic state with autophagy regulation. We thus propose C9orf72 as a fundamental component of autophagy signaling with implications in basic cell physiology and pathophysiology, including neurodegeneration. KEYWORDS ALS; autophagy; C9orf72; FTD; lysosome; MTOR; neurodegeneration; p62; SMCR8; TFEB A hexanucleotide repeat expansion in the promoter or intronic region of isoforms of the C9orf72 gene is responsible for the most common forms of the neurodegenerative diseases ALS and FTD, as well as rare cases of Alzheimer, Parkinson, Huntington, and other diseases. While the repeat expansion reduces the level of C9orf72 protein in patients, and insufficiency in protein function has been proposed to contribute to pathogenesis, the functions of the C9orf72 protein are poorly understood. C9orf72 is predicted to be a member of the DENN domain protein superfamily, whose members have been involved in RAB-mediated membrane trafficking pathways. Using animal, cell, and proteomic tools to study the function of the C9orf72 protein we report the surprising finding that loss of C9orf72 results in enhanced autophagic activity, under both basal and starvation conditions. Thus, we propose C9orf72 as a negative regulator of autophagy (Fig. 1).We generated 3110043O21Rik/C9orf72 knockout mice and observed a late-onset age-dependent lethality phenotype in the homozygote, and to a lesser degree, the heterozygous animals. The cause of the animal death remains unclear, although immune dysregulation could be involved. To investigate the molecular functions of C9orf72 we used human and mouse knockdown/knockout cells, and determined that loss of the C9orf72 protein systemically perturbs autophagy regulation. We revealed an impairment to MTOR signaling because MTOR activation after amino acid stimulation is diminished in the absence of C9orf72. In addition, we observed an enhancement of TFEB (transcription factor EB) signaling: Loss of C9orf72 increases TFEB protein levels and promotes its nuclear translocation.Consistent with inhibited MTOR and upregu...

Stable transgenic C9orf72 zebrafish model key aspects of the ALS/FTD phenotype and reveal novel pathological features

“…Our data are consistent with several other studies in animal models showing toxicity mediated by RNA foci and DPRs [ 5 , 20 , 26 , 38 ], including two independently generated C9orf72 zebrafish models [ 18 , 28 ]. Furthermore, our data are consistent with four independently generated C9orf72 knock-out mice and one knockout zebrafish model, none of which display any motor or neurodegenerative changes, arguing against haploinsufficiency as a major contributor to C9orf72 ALS/FTD [ 1 , 13 , 17 , 35 ], (Schmid, Hruscha, Haass, unpublished). In contrast, decreased C9orf72 transcript levels have been reported in the CNS of G 4 C 2 expansion bearing patients, and morpholino mediated knockdown of C9orf72 transcripts have been linked with motor deficits in zebrafish [ 6 , 11 ].…”

“…However, early reports from C9orf72 knockout zebrafish do not recapitulate the knockdown motor phenotypes ([ 34 ]; Schmid, Hruscha, Haass, unpublished). Additionally, four independently generated C9orf72 knockout mice did not demonstrate any neurodegenerative phenotype [ 1 , 13 , 17 , 35 ].…”

TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS

“…Regulation of protein and lipid homeostasis by the lysosome may be particularly important in neurons since they are post-mitotic and have high energy demands (Fraldi, Klein, Medina, & Settembre, 2016). Loss of function of C9ORF72 also disrupts 10 autophagy and lysosomal function in multiple cell types (Farg et al, 2014;Ji, Ugolino, Brady, Hamacher-Brady, & Wang, 2017;O'Rourke et al, 2015;Sellier et al, 2016;Y. Shi et al, 2018;Sullivan et al, 2016;Ugolino et al, 2016;Webster et al, 2016;Yang et al, 2016;Zhu et al, 2020), suggesting a mechanism whereby G4C2 repeats may have synergistically detrimental effects with haploinsufficient C9ORF72 in C9-ALS/FTD patients.…”

Association Between Autophagy and Neurodegenerative Diseases

“…Mutations in C9orf72 , the most common cause of ALS, have been proposed to downregulate autophagy (Ji et al, 2017 ). C9orf72 is a core component of GDP/ GTP exchange factor (GEF) for Rab8 and Rab39, which is crucial for autophagosome maturation.…”